Mass Spectrometry (MS)-based quantitative proteomics is a robust technology for the systematic understanding of biological processes. Several MS-based methods, using either stable isotope-labeling or label-free strategies, have been developed to improve the throughput and accuracy of protein quantitation. The stable-isotope labeling methods rely on the introduction of stable isotope tags to proteins, and the labeled proteins can be distinguished and quantified by MS. Label-free quantitation is performed by spectral count and statistical analysis of unlabeled proteolytic peptides. Although label-free methods are implemented without incurring additional reagent costs, isotope label-based strategies can provide more accurate quantitation and capabilities of processing multiple specimens in parallel. This has been demonstrated by commercially available 8-plex iTRAQ (Isobaric Tag for Relative and Absolute Quantitation) and 6-plex TMT (Tandem Mass Tag) reagents.
Stable isotope labeling methods, such as ICAT (Isotope Coded Affinity Tag), iTRAQ (Isobaric Tag for Relative and Absolute Quantitation), and SILAC (Stable Isotope Labeling with Amino Acids in Cell Culture), are widely used for the quantitative comparison of proteins, providing versatile tools for proteomics research and biomarker discovery. (Ong, S. E.; Mann, M. Nat Chem. Biol. 2005, 1, 252-62; Yan, W.; Chen, S. S. Brief Funct. Genomic Proteomic 2005, 4, 27-38; Julka, S.; Regnier, F. E. Brief Funct. Genomic Proteomic 2005, 4, 158-77.)
Known isotope labeling methods use reagents that are coded with common isotope pairs, including 2H/1H, 13C/12C, and 15N/14N, to label identical peptides or proteins to make them distinguishable by MS. (Ong, S. E.; Blagoev, B.; Kratchmarova, I.; Kristensen, D. B.; Steen, H.; Pandey, A.; Mann, M. Mol Cell Proteomics 2002, 1, 376-86; Ross, P. L. et al. Mol Cell Proteomics 2004, 3, 1154-69; Li, S.; Zeng, D. Chem. Commun. 2007, 2181-3; Wiese, S.; Reidegeld, K. A.; Meyer, H. E.; Warscheid, B. Proteomics 2007, 7, 340-50.)
Unfortunately, known reagents are prohibitively expensive (for example, iTRAQ 8-plex 1-assay kit from Applied Biosystems, $595.00 USD; TMT 6-plex 5-assay kit from Thermo Fisher Scientific Inc, $2300.00 USD), limiting their use in basic biological studies and clinical applications where a large number of samples need to be processed. The high costs of these reagents are due, in part, to the fact that they are coded with expensive 13C, 15N or 18O, but not with the less expensive 2H. Additionally, the preparation of iTRAQ and TMT reagents is quite complicated and requires expensive starting materials. For example, the synthesis of TMT reagents involves a daunting number of 14 steps with an overall yield less than 1%.
Though 2H-labeled molecules were developed as the prototype reagents in the first generation isotope labeling technologies (Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R. Nat Biotechnol 1999, 17, 994-9),they have been largely phased out and replaced by 13C— or 15N-coded reagents because these early generation 2H coded reagents cause chromatographic shift in reverse phase HPLC and compromise the accuracy of quantitation by LC-MS/MS. (Yi, E. C.; Li, X. J.; Cooke, K.; Lee, H.; Raught, B.; Page, A.; Aneliunas, V.; Hieter, P.; Goodlett, D. R.; Aebersold, R. Proteomics 2005, 5, 380-7.)
However, as 2H-labeled compounds are usually easier and less expensive to synthesize than their 13C— or 15N-coded counterparts, it is still of great interest to develop 2H-based technologies for protein quantitation, if 2H-related chromatographic shift could be eliminated. Recently, a study to identify structural features of 2H-containing molecules that are responsible for their isotope effects has found that placing 2H atoms next to hydrophilic groups and minimizing the number of 2H atoms in a molecule can reduce their contribution to isotope effects, providing useful clues for the design of 2H-based tags that are irresolvable by HPLC. (Zhang, R.; Sioma, C. S.; Thompson, R. A.; Xiong, L.; Regnier, F. E. Anal Chem 2002, 74, 3662-9.)
There therefore remains a need in the art to develop 2H-based reagents and products containing such reagents for biomolecular quantitation. Cost effective and low complexity methods of making such reagents are also needed.